Inhibition of RhoA/Rho-kinase pathway suppresses the expression of extracellular matrix induced by CTGF or TGF-β in ARPE-19

AIM:To investigate the role of Rho-associated protein kinase (ROCK) inhibitor, Y27632, in mediating the production of extracellular matrix (ECM) components including fibronectin, matrix metallo-proteinase-2 (MMP-2) and type I collagen as induced by connective tissue growth factor(CTGF) or transforming growth factor-β (TGF-β) in a human retinal pigment epithelial cell line, ARPE-19. METHODS:The effect of Y27632 on the CTGF or TGF-β induced phenotype in ARPE-19 cells was measured with immunocytochemistry as the change in F-actin. ARPE-19 cells were treated with CTGF (1, 10, 100ng/mL)and TGF-β (10ng/mL) in serum free media, and analyzed for fibronectin, laminin, and MMP-2 and type I collagen by RT-qPCR and immunocytochemistry. Cells were also pretreated with an ROCK inhibitor, Y27632, to analyze the signaling contributing to ECM production. ·RESULTS:Treatment of ARPE-19 cells in culture with TGF-β or CTGF induced an ECM change from a cobblestone morphology to a more elongated swirl pattern indicating a mesenchymal phenotype. RT-qPCR analysis and different gene expression analysis demonstrated an upregulation in expression of genes associated with cytoskeletal structure and motility. CTGFor TGF-β significantly increased expression of fibronectin mRNA (P =0.006, P =0.003 respectively), laminin mRNA (P =0.006, P =0.005), MMP-2 mRNA (P =0.006, P =0.001), COL1A1 mRNA (P =0.001, P =0.001), COL1A2 mRNA (P = 0.001, P =0.001). Preincubation of ARPE-19 with Y27632 (10mmol/L) significantly prevented CTGF or TGF-β induced fibronectin (P=0.005, P=0.003 respectively), MMP-2 (P = 0.003, P =0.002), COL1A1 (P =0.006, P =0.003), and COL1A2 (P =0.006, P =0.004) gene expression, but not laminin (P =0.375, P =0.516). CONCLUSION:Our study demonstrated that both TGF-β and CTGF upregulate the expression of ECM components including fibronectin, laminin, MMP-2 and type I collagen by activating the RhoA/ROCK signaling pathway. During this process, ARPE-19 cells were shown to change from an epithelial to a mesenchymal phenotype in vitro. Y27632, a ROCK inhibitor, inhibited the transcription of fibronectin, MMP-2 and type I collagen, but not laminin. The data from our work suggest a role for CTGF as a profibrotic mediator. Inhibiting the RhoA/ROCK pathway represents a potential target to prevent the fibrosis of retinal pigment epithelial (RPE) cells. This might lead to a novel therapeutic approach to preventing the onset of early proliferative vitreoretinopathy(PVR).

Centralized isolation of Helicobacter pylori from multiple centers and transport condition influences

AIM:To evaluate the efficacy of centralized culture and possible influencing factors.METHODS:From January 2010 to July 2012,66452 patients with suspected Helicobacter pylori(H.pylori) infection from 26 hospitals in Zhejiang and Jiangsu Provinces in China underwent gastrointestinal endoscopy.Gastric mucosal biopsies were taken from the antrum for culture.These biopsies were transported under natural environmental temperature to the central laboratory in Hangzhou city and divided into three groups based on their transport time:5,24 and 48 h.The culture results were reported after 72 h and the positive culture rates were analyzed by a χ2 test.An additional 5736 biopsies from H.pylori-positive patients(5646 rapid urease test-positive and 90 14C-urease breath test-positive) were also cultured for quality control in the central laboratory setting.RESULTS:The positive culture rate was 31.66%(21036/66452) for the patient samples and 71.72%(4114/5736) for the H.pylori-positive quality control specimens.In the 5 h transport group,the positiveculture rate was 30.99%(3865/12471),and 32.84%(14960/45553) in the 24 h transport group.In contrast,the positive culture rate declined significantly in the 48 h transport group(26.25%; P < 0.001).During transportation,the average natural temperature increased from 4.67 to 29.14℃,while the positive culture rate declined from 36.67%(1462/3987) to 24.12%(1799/7459).When the temperature exceeded 24℃,the positive culture rate decreased significantly,especially in the 48 h transport group(23.17%).CONCLUSION:Transportation of specimens within 24 h and below 24℃ is reasonable and acceptable for centralized culture of multicenter H.pylori samples.

Extracting Campus’Road Network from Walking GPS Trajectories

Road network extraction is vital to both vehicle navigation and road planning.Existing approaches focus on mining urban trunk roads from GPS trajectories of floating cars.However,path extraction,which plays an important role in earthquake relief and village tour,is always ignored.Addressing this issue,we propose a novel approach of extracting campus’road network from walking GPS trajectories.It consists of data preprocessing and road centerline generation.The patrolling GPS trajectories,collected at Hunan University of Science and Technology,were used as the experimental data.The experimental evaluation results show that our approach is able to effectively and accurately extract both campus’trunk roads and paths.The coverage rate is 96.21%while the error rate is 3.26%.

Limbal epithelial stem cells in corneal surface reconstruction

Cornea serves as the partial front barrier and major light reflection organ of the eye.The integrity of corneal surface is essential for ocular function.Injuries or congenital diseases could significantly destruct the homeostasis of the ocular surface,especially the microenvironment of limbal epithelial stem cells(LESCs),and will eventually cause dysfunction of corneal regeneration and diminish of LESCs.The loss of LESCs by different reasons are named limbal stem cell deficiency(LSCD),which is one of the leading cause of vision loss worldwide.To restore the corneal surface,LESC transplantation in the form of tissue or cell cultures is currently a viable and promising method to treat LSCD.In this review,we aim to introduce the characters and niche of LESCs,and discuss different aspects of its application in cornea surface reconstruction.

Lipid metabolism dysfunction induced by age-dependent DNA methylation accelerates aging

Epigenetic alterations and metabolic dysfunction are two hallmarks of aging.However,the mechanism of how their interaction regulates aging,particularly in mammals,remains largely unknown.Here we show ELOVL fatty acid elongase 2(Elovl2),a gene whose epigenetic alterations are most highly correlated with age prediction,contributes to aging by regulating lipid metabolism.We applied artificial intelligence to predict the protein structure of ELOVL2 and the interaction with its substrate.Impaired Elovl2 function disturbs lipid synthesis with increased endoplasmic reticulum stress and mitochondrial dysfunction,leading to key aging phenotypes at both cellular and physiological level.Furthermore,restoration of mitochondrial activity can rescue age-related macular degeneration(AMD)phenotypes induced by Elovl2 deficiency in human retinal pigmental epithelial(RPE)cells;this indicates a conservative mechanism in both human and mouse.Taken together,we revealed an epigenetic-metabolism axis contributing to aging and illustrate the power of an AI-based approach in structure-function studies.

D609 protects retinal pigmented epithelium as a potential therapy for age-related macular degeneration

Accumulated oxidative damage may lead to irreversible retinal pigmented epithelium(RPE)cell death,which is considered to be the primary cause of dry age-related macular degeneration(AMD),leading to blindness in the elderly.However,an effective therapy for this disease is lacking.Here,we described a robust high-content screening procedure with a library of 814 protective compounds and found that D609 strongly protected RPE cells from sodium iodate(SI)-induced oxidative cell death and prolonged their healthy survival.D609 effectively attenuated excessive reactive oxygen species(ROS)and prevented severe mitochondrial loss due to oxidative stress in the RPE cells.Surprisingly,the potent antioxidative effects of D609 were not achieved through its own reducibility but were primarily dependent on its ability to increase the expression of metallothionein.The injection of this small water-soluble molecule also showed an explicit protective effect of the RPE layer in an SI-induced AMD mouse model.These findings suggested that D609 could serve as a novel antioxidative protector of RPE cells both in vitro and in vivo and unveiled a novel antioxidative mechanism of D609,which may ultimately have clinical applications for the treatment of AMD.

Loss of FOXC1 contributes to the corneal epithelial fate switch and pathogenesis

Forkhead box C1(FOXC1)is required for neural crest and ocular development,and mutations in FOXC1 lead to inherited Axenfeld-Rieger syndrome.Here,we find that FOXC1 and paired box 6(PAX6)are co-expressed in the human limbus and central corneal epithelium.Deficiency of FOXC1 and alternation in epithelial features occur in patients with corneal ulcers.FOXC1 governs the fate of the corneal epithelium by directly binding to lineage-specific open promoters or enhancers marked by H3K4me2.FOXC1 depletion not only activates the keratinization pathway and reprograms corneal epithelial cells into skin-like epithelial cells,but also disrupts the collagen metabolic process and interferon signaling pathways.Loss of interferon regulatory factor 1 and PAX6 induced by FOXC1 dysfunction is linked to the corneal ulcer.Collectively,our results reveal a FOXC1.mediated regulatory network responsible for corneal epithelial homeostasis and provide a potential therapeutic target for corneal ulcer.